1189DEVELOPMENT AND DISEASE RESEARCH ARTICLE

INTRODUCTIONAmyloid precursor protein (APP) is a transmembrane protein thatplays a key role in Alzheimer’s disease. This disease ischaracterized by intraneuronal tangles and extracellular plaques,and the amyloid beta-peptide (A�), which is derived from APPupon cleavage by �- and �-secretases, is the major component ofthe plaques. Mutations in APP have been linked to familialAlzheimer’s disease, and the most widely accepted models ofdisease etiology propose that A� aggregates or oligomers triggera cascade of events causing damage to neuronal connections andcell death (Selkoe, 1999; Hardy and Selkoe, 2002; Tanzi andBertram, 2005; Catalano et al., 2006).

The functions of APP in normal physiology and developmentare not well understood. APP-deficient mice are viable and fertile,but have some abnormalities, including susceptibility to seizures(Steinbach et al., 1998) and a defect in corpus callosum formation(Magara et al., 1999), indicating roles in neural development andfunction. Triple knockout mice for APP and its two homologs,APLP1 (amyloid precursor like protein 1) and APLP2, exhibit acortical defect reminiscent of human type 2 lissencephaly,suggesting a role in neuronal migration, and die perinatally,although the exact reasons for this remain mysterious (Herms etal., 2004).

Since its initial identification, APP was thought likely to be areceptor, based on its transmembrane structure (Kang et al.,1987). By analogy to Notch (Mumm and Kopan, 2000), an APPsignaling pathway has been proposed where �-secretase cleavageyields the C-terminal CTF� fragment, then �-cleavage liberatesthe APP intracellular domain to participate in downstreampathways (Chan and Jan, 1998). Some evidence has accumulatedto support this model of signaling (Cao and Sudhof, 2001;Kimberly et al., 2001; Kinoshita et al., 2002), although someaspects remain controversial (Cao and Sudhof, 2004; Hass and

Yankner, 2005; Hebert et al., 2006). Several cytoplasmic bindingpartners for APP have been identified, delineating potentialdownstream pathways (Kerr and Small, 2005). However, the keysignaling mechanisms remain unclear, especially as it is not clearwhich, if any, of the identified extracellular binding partners forAPP might function as a physiological ligand.

In addition to the potential receptor function of APP, its cleavedectodomain, APPs�, may function as a ligand. Numerous studieshave shown that APPs� can modulate cell behaviors includingneurite outgrowth, synaptogenesis, neurogenesis and cell survivaland proliferation (Mattson, 1997; Turner et al., 2003; Caille et al.,2004). Distinct domains of APPs� have been implicated in theseactions, suggesting the existence of more than one receptor. To date,however, no cell-surface receptor capable of mediating APPs�-induced signaling has been identified.

More than a decade of work has led to the identification of anumber of extracellular partners that can interact with APP,directly or indirectly. Binding has been reported for extracellularmatrix components, including heparan sulfate (Multhaup, 1994;Small et al., 1994), collagen (Beher et al., 1996) and fibulin 1(Ohsawa et al., 2001); zinc and copper ions (Turner et al., 2003);and the lipoprotein receptors, scavenger receptor A (Santiago-Garcia et al., 2001) and LRP (Kounnas et al., 1995). Morerecently identified extracellular proteins that can interact withAPP include F-spondin (also known as spondin 1) (Ho andSudhof, 2004; Hoe et al., 2005), Drosophila FASII (Ashley et al.,2005), BRI2 (ITM2B) (Fotinopoulou et al., 2005; Matsuda et al.,2005), APLP1, APLP2 and APP itself (Soba et al., 2005), Notchfamily members (Fassa et al., 2005; Fischer et al., 2005; Oh et al.,2005; Chen et al., 2006), LRRTM3 (Majercak et al., 2006) andNgR (RTN4R) (Park et al., 2006). Some of these proteins caninfluence candidate downstream signaling pathways or APPprocessing. However, these interactions have generally not yetbeen characterized thoroughly with regard to whether they haveaffinity and specificity in the range of cognate receptor-ligandinteractions, involve direct interaction with APP, and whetherthey can affect cell behavior. There is also generally littleevidence regarding potential roles for these interactions invertebrate neural development.

Interaction of amyloid precursor protein with contactins andNgCAM in the retinotectal systemMiriam Osterfield, Rikke Egelund, Lauren M. Young and John G. Flanagan*

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease, but its actions in normal development are not wellunderstood. Here, a tagged APP ectodomain was used to identify extracellular binding partners in developing chick brain.Prominent binding sites were seen in the olfactory bulb and on retinal axons growing into the optic tectum. Co-precipitation fromthese tissues and tandem mass spectrometry led to the identification of two associated proteins: contactin 4 and NgCAM. In vitrobinding studies revealed direct interactions among multiple members of the APP and contactin protein families. Levels of the APPprocessing fragment, CTF�, were modulated by both contactin 4 and NgCAM. In the developing retinotectal system, APP, contactin4 and NgCAM are expressed in the retina and tectum in suitable locations to interact. Functional assays revealed regulatory effectsof both APP and contactin 4 on NgCAM-dependent growth of cultured retinal axons, demonstrating specific functional interactionsamong these proteins. These studies identify novel binding and functional interactions among proteins of the APP, contactin andL1CAM families, with general implications for mechanisms of APP action in neural development and disease.

KEY WORDS: Amyloid precursor protein, Axon, Contactin, L1CAM, Retina

Development 135, 1189-1199 (2008) doi:10.1242/dev.007401

Department of Cell Biology and Program in Neuroscience, Harvard Medical School,Boston, MA 02115, USA.

*Author for correspondence (e-mail: flanagan@hms.harvard.edu)

Accepted 9 January 2008 DEVELO

PMENT

1190

Contactins, which are GPI-anchored, and L1CAM familyproteins, which are transmembrane proteins, are members of theIgCAM (immunoglobulin-related cell adhesion molecule)superfamily. Contactins and L1CAMs can bind one another, as wellas partially overlapping sets of other cell-surface or extracellular-matrix proteins (Sonderegger, 1997). L1CAM and its chickenhomolog NgCAM have been widely studied for functions in axongrowth, guidance and fasciculation (Hortsch, 1996; Sonderegger,1997; Maness and Schachner, 2007), as have contactins 1 and 2(Falk et al., 2002). Contactins 3 (PANG/BIG-1) and 4 (BIG-2) areless well characterized, although both can promote neuriteoutgrowth (Connelly et al., 1994; Yoshihara et al., 1994; Yoshiharaet al., 1995). Like APP, contactin 4 and L1CAM are implicated inneurological disorders. Contactin 4 gene disruption is proposed tocause 3p deletion syndrome, involving mental retardation(Fernandez et al., 2004; Dijkhuizen et al., 2006). Mutations inL1CAM cause CRASH syndrome, which includes mentalretardation and corpus callosum hypoplasia (Fransen et al., 1995).

Here, we took the approach of initially testing embryonic chickbrain for APP-binding sites, and found particularly prominentbinding on retinal axons growing into the optic tectum, a well-characterized model of axon development (McLaughlin andO’Leary, 2005; Flanagan, 2006). We next identified extracellularbinding partners and found APP to have a direct high affinityinteraction with contactins 3 and 4. APP also associates, directly orindirectly, with tectal NgCAM. APP, contactins and NgCAM are allexpressed in the retinotectal system. In functional assays of culturedretinal ganglion cells (RGCs), contactin 4 and APP modulated axonbehavior specifically in the context of NgCAM-dependent axongrowth, demonstrating functional interactions among these proteins.Our studies of binding, expression and functional effects on cellbehavior identify novel interactions of APP, with generalimplications for development and disease.

MATERIALS AND METHODSDNA constructsAP-APPs� contains amino acids 18-612 of APP695; human in Fig. 4, mouseelsewhere. AP-APLP1 encodes residues 34-567 of human APLP1(NM_005166). N-terminal alkaline phosphatase (AP) fusion constructs werein APtag4 (Flanagan et al., 2000). APP-HA contains full-length humanAPP695, then an HA tag, in pcDNA3.1 Zeo+. A start codon was placedupstream of APP amino acid 597 for CTF�, and 613 for CTF�.

NgCAM-Fc encodes residues 1-1134 from pSCT-NgCAM, kindlyprovided by P. Sonderegger (Buchstaller et al., 1996), in pSecTagIg. Fusionsof all six human contactins were full length up to the GPI anchor site,followed by Fc, in IgTag2Eco. Accession numbers and predicted GPI sitesare: contactin 1 (NM_001843, 993); 2 (NM_005076, 1012); 3 (NM_020872,1002); 4 (NM_175607, 1000); 5 (NM_014361, 1072); 6 (NM_014461,999). Deletion constructs encode the following regions in pSecTagIg: c3Ig1-4 (20-404), c3Ig5-6 (405-597), c3FN (598-1005), c4Ig1-4 (19-404), c4Ig5-6 (405-595), c4FN (596-1000). Contactin 4-AP contains residues 1-1000,then C-terminal AP, in APtag2. Chick contactin 4-myc-His contains residues1-1005 in pcDNA3.1/myc-His.

Chick in situ probes in pBluescript II SK(–) contain ORF nucleotides:APP (AF289218), 471-1218; NgCAM (Z75013), 2827-3759; contactin 3(NM_414433), 2465-3277; contactin 4 (XM_414435), 418-1212.

Immunolocalization, RNA and affinity probe in situsAP fusion proteins were used as in situ probes as described (Flanagan et al.,2000). In situ hybridization used 10-�m sections of E11.5 chick with detectionby AP-coupled anti-digoxigenin (Roche) or, for fluorescent in situs,peroxidase-conjugated anti-digoxigenin antibody (Roche) then Alexa Fluor488 with tyramide amplification (Molecular Probes). Immunolocalizationused rabbit anti-GAP43 (Novus Biologicals), or goat anti-APP (44-63)(Calbiochem), then fluorescent secondary antibodies (Molecular Probes).

AP and Fc fusion proteinsFusion proteins were produced in transiently transfected 293T cells(Flanagan et al., 2000). For axon growth experiments, fusion proteins wereproduced in Opti-MEM plus ITS-A (Invitrogen), then AP fusions wereconcentrated 10-fold using Amicon Centricon (Millipore), or Fc fusionswere purified with Protein A-Sepharose beads (4 Fast Flow, Amersham).

Protein purifications from brainFor purification of APP-binding proteins, tecta from E12.5 chick embryos(approximately 100 for small-scale experiments, 1200 for large-scale) weretreated as follows, at room temperature unless indicated: Hank’s bufferedsalt solution (HBSS) wash; 0.2 mg/ml EZ-link NHS-LC-Biotin (Pierce) inHH (HBSS with 20 mM HEPES, pH 7.0), 45 minutes; 50 mM Tris pH 7.5in HH, 20 minutes; HBAH (HBSS with 0.5 mg/ml BSA, 0.05% sodiumazide and 20 mM HEPES, pH 7.0) wash; AP fusion-containing conditionedmedia, at least 90 minutes; ice-cold HBAH, six washes; ice-cold HBSS, sixwashes; 0.5 mg/ml DTSSP (Pierce) in HH, 45 minutes; 50 mM Tris pH 7.5in HH, 20 minutes; Lysis buffer (0.5% sodium deoxycholate, 0.5% TritonX-114 and 0.05% SDS in PBS) plus protease inhibitors (5 mM EDTA pluscOmplete, Roche), 45 minutes, ice. Supernatant was cleared 100,000 g, 4°C,then incubated overnight at 4°C with anti-AP (MIA1801, Seradyn) beads asdescribed (Flanagan et al., 2000). Beads were washed with ice-cold Lysisbuffer, then Wash buffer (0.1% Triton X-100 in PBS), eluted with 100 mMglycine, 150 mM NaCl, pH 3.0, then buffered with 100 mM Tris pH 8.0. ForSDS-PAGE, proteins were precipitated with 10% TCA. For 2D gels, samplewas boiled 15 minutes in 100 mM DTT, then incubated with 9.25 mg/mliodoacetamide, 15 minutes, before TCA precipitation.

For purification of tectal surface proteins, 800 E12.5 chick tecta wereincubated in HBSS (one wash); 1 mg/ml Sulfo-NHS-SS-Biotin (Pierce) inHH, 1 hour; 50 mM Tris pH 7.5 in HH, 20 minutes; then HBSS (severalwashes). Tectal lysis and clearing were as above, followed by overnightincubation at 4°C with NeutrAvidin beads (Pierce). Beads were washed withthe following buffers: Lysis, Wash, High salt wash (1 M NaCl, 20 mM HEPESpH 7.0 and 0.1% Triton X-100), then Wash. The biotinylation reagent wascleaved in 100 mM DTT (65°C, 40 minutes), then protein was TCAprecipitated.

The ZOOM IPGRunner system (Invitrogen) was used for 2D gels, usingpH 3-10 strips and 4-20% Tris-glycine gels. Sample buffer was from the 2DInsoluble Protein Sample Prep Kit (Pierce) with carrier ampholytes(Invitrogen), 20 mM DTT and 0.02 mg/ml bromophenol blue. Silverstaining was as described (Shevchenko et al., 1996). For western blots, biotinwas detected with horseradish peroxidase-conjugated NeutrAvidin (Pierce).

Antibodies to test candidate tectal proteins were anti-PSA-NCAM(Amersham), anti-chick NCAM-1 (Amersham), anti-tenascin (Chemicon), anti-neogenin (R&D systems), anti-contactin 1 (BD Biosciences) and three chickNgCAM antibodies: mouse monoclonal H23, kindly provided by J. Sanes(Yamagata et al., 1995) and mouse monoclonal mAb 12-I-4E-311 and rabbitpolyclonal rb 4003, both kindly provided by F. Rathjen (Chang et al., 1990).

Binding and APP processing assaysBinding assays using fusion proteins were as described (Flanagan andCheng, 2000). AP fusions were normalized for AP activity. Fc fusions werequantitated by anti-Fc western with Li-COR Odyssey scanner. For Fig. 5A,relative concentration of Fc fusion proteins, as compared with contactin 3FN-Fc, were: c3Ig1-4-Fc, 2.7; c3Ig5-6-Fc, 0.2; c3FN-Fc, 1; c4Ig1-4-Fc, 0.1;c4Ig5-6-Fc, 5.5; c4FN-Fc, 1.7. For Fig. 4A, Fc concentrations werenormalized.

For APP processing assays, 293T cells were transfected using TransIT-LT1 (Mirus Bio). After 1-2 days, plates were ice chilled, 5 minutes; lysed in1% Triton X-100, 10 mM Tris pH 8.0, plus 140 mM NaCl, 20 minutes; andcleared lysates were combined with sample buffer. Western blots wereprobed with anti-HA (monoclonal 3F10, Roche), followed by HRP- orIRdye800-conjugated anti-rat antibody (Rockland).

RNAi experimentsControl virus contains EGFP-F (Clontech) in RCASBP(B) replication-competent retrovirus. For contactin 4 shRNA virus, pGSU6-GFP (Genlantis)was used according to manufacturer’s instructions with target sequence

RESEARCH ARTICLE Development 135 (6)

DEVELO

PMENT

CTACGAGTGTGTCGCTGAA (Reynolds et al., 2004), and the U6promoter and shRNA sequence were inserted into the control virusdownstream of EGFP-F. Because we do not have an antibody to chickcontactin 4, knockdown was tested on tagged chick contactin 4-myc-His,co-transfected with contactin 5-Fc control, APtag5 and shRNA or controlRCASBP(B) plasmid into 293T cells. Supernatants, normalized fortransfection by AP activity, were analyzed by western blot with IRdye800-conjugated anti-myc and IRdye700DX-conjugated anti-human Fc. TheshRNA construct, compared with control virus, was confirmed tospecifically knockdown protein levels of contactin 4 (P<0.0001), and notthe contactin 5 internal control (see Fig. S1D in the supplementary material).For outgrowth assays, stage 9-11 chick embryos were electroporated withRCASBP(B) plasmids as described (Schulte et al., 1999). Greenfluorescence was used to select highly infected (approximately 50-95%)retinas.

RGC axon growth assaysStage 27-28 chick retinas were mounted on polycarbonate filter (Sartorius),cut into 300 �m strips, and cultured RGC-side down for 2 days in 47.6%Neurobasal media, 37.5% DMEM/F12, 4.8% FBS, 2.4% chick serum, 1%B27, 0.15% methyl cellulose, 0.14% glucose, 20 mM HEPES pH 7.0, Pen-Strep and 1.76 �g/ml glutamate. Supernatant from one homogenized stage27-28 chick brain was added per 5 ml media.

Glass coverslips were first coated with 20 �g/ml poly-L-lysine. Wheremultiple purified proteins (Fc fusions, 20 �g/ml; or laminin, 10 �g /ml, BDBiosciences) were used, they were mixed to coat coverslips simultaneously.For purified proteins plus a supernatant (AP fusion or mock), coating wasfirst with purified proteins, then supernatants. Axons were fluorescentlylabeled in 33 �M carboxyfluorescein diacetate, succinimidyl ester(Invitrogen), 10 minutes. Since basal growth varied between experiments,most figures show a single experiment, with similar trends being observedin multiple experiments, except Fig. 7K which shows three experimentsnormalized to the average growth of control axons on NgCAM+APPs�.

RESULTSLocalization of two distinct APP-binding partnersin embryonic chick brainTo search for novel extracellular binding partners for APP, wegenerated an alkaline phosphatase (AP) fusion protein, AP-APPs�,for use as an affinity probe (Flanagan and Cheng, 2000). We foundthat AP-APPs� binds to embryonic chick brain, with particularlyprominent binding to the optic tectum (Fig. 1B). Binding was alsoseen in the olfactory bulb, as well as other regions (Fig. 1B). Withinthe tectum, the most prominent AP-APPs� binding appeared to belocalized to RGC axons. The staining spread across the tectum witha developmental time course similar to RGC axon ingrowth(Goldberg, 1974), appearing at the anterior end by Hamburger-

Hamilton (HH) stage 33 (E8.5 under our conditions), with somelabeled axons reaching the posterior tectum by HH 38 (E12.5), andcovering the tectum by HH 41 (E15.5) (Fig. 1D-F and data notshown). The staining also had a prominently striated appearance thatis characteristic of axons (Fig. 1C). Furthermore, in monocular chickembryos (one naturally occurring, two surgically enucleated), AP-APPs� binding was strongly reduced in the tectal hemispherecontralateral to (and therefore normally innervated by) the missingeye (Fig. 1G).

Two lines of evidence suggested that AP-APPs� was detectingmore than one binding partner. First, a nested deletion analysis ofAPP identified two regions that produced different binding patterns(Fig. 2A). A middle domain (amino acids 199-293) was sufficientfor weak binding to RGC axons in the tectum (Fig. 2C), whereas asomewhat longer region (amino acids 199-345) gave stronger RGCbinding comparable to full-length AP-APPs� (Fig. 2B; bindingpattern 1; green bar in Fig. 2A), but both gave little binding abovebackground to the olfactory bulb (Fig. 2B,C). By contrast, an N-terminal domain of APP (amino acids 18-205) showed particularlystrong binding to the olfactory bulb, in addition to widespreadbinding above background throughout the brain, including thetectum (Fig. 2F; binding pattern 2; blue bar in 2A). Second,treatment of brains with PI-PLC (phosphatidylinositol-specificphospholipase C), which cleaves GPI anchorages, greatly reducedAP-APPs� binding to the olfactory bulb, whereas binding to tectaappeared less affected (Fig. 2D,E), suggesting that the predominantAPP binding partner detected in olfactory bulbs is GPI-anchored,whereas the tectum appears to contain a GPI-anchored bindingpartner and additional non-GPI-anchored partner(s).

The APLP1 ectodomain, fused to an AP tag, was also tested forbinding to embryonic chick brain. Binding was widespread, butparticularly prominent in the olfactory bulb (Fig. 2G), similar to thatobserved for the APP N-terminal domain, suggesting that the N-terminal domains of APP and APLP1 might share binding partners(consistent with our subsequent molecular experiments).

APP binds specific contactinsWe next developed a protocol to selectively purify extracellularbinding partners for APP from sites in developing brain withprominent APP binding. Tecta were surface biotinylated, thenincubated in one of three probes (AP-APPs�, AP-APP(199-345) orAP control), washed, incubated in DTSSP (a cleavable, membrane-impermeable crosslinker) then lysed and immunoprecipitated for theAP tag. Subsequent western blots for biotin revealed prominentbands that co-immunoprecipitated with AP-APPs� but not with AP

1191RESEARCH ARTICLEAPP interaction with contactins and NgCAM

Fig. 1. APP-binding sites in embryonic chickbrain. (A,B) Binding of AP-APPs� versus AP control.Ventral view, E11.5-12.5 brains. Prominent bindingin optic tectum (OT, arrow), with additional stainingin olfactory bulbs (OB, arrowhead) and other regions.(C) AP-APPs� staining of tectum at greatermagnification, showing anterior-posterior striationcharacteristic of axons in the stratum opticum.Anterior-posterior is horizontal. Scale bar: 100 �m.(D-F) Time-course of AP-APPs� binding to brains.Dorsal view. Anterior (A) and posterior (P) extremesof tecta. Arrows indicate posterior limit of staining.(G) Binding of AP-APPs� to brain from embryo withsingle enucleated eye. Ventral view. Staining incontralateral tectum (arrow) is reduced tobackground levels. AP, alkaline phosphatase. D

EVELO

PMENT

1192

alone (Fig. 3A). AP-APP(199-345) co-immunoprecipitated thesame biotinylated bands, though to a lesser degree (data not shown).Two prominent bands between 120 kDa and 150 kDa were seen inrepeated experiments (Fig. 3A, asterisks). Additional, fainter bandsat higher molecular weight were sometimes seen.

To identify these bands, we scaled up the above purification. Eachco-immunoprecipitation (co-IP) was run on a 2D gel and silverstained (Fig. 3C,D,F); the AP-tagged probes alone were runseparately for comparison (Fig. 3G and data not shown). A separategel with a western blot for biotin was used as an additional guide todetermine which region of the silver-stained gels to analyze (Fig.3B,E). Because the signal was not intense, we pooled the samplesfrom the AP-APPs� co-IP and AP-APP(199-345) co-IP gels, so anyproteins identified might interact with AP-APP(199-345), AP-APPs�, or both. Tandem mass spectrometry identified humanplacental alkaline phosphatase and APP, both presumably derivedfrom the probe, and only one other protein, contactin 4, as a strongmatch to the sample. Contactin 4 was also among the proteins weidentified in an independent approach involving purification andmass spectrometry of GPI-anchored proteins in the olfactory bulb(data not shown).

These mass spectrometry studies suggested that contactin 4 bindsAPP. However, interactions in the context of an intact cell or tissuecan be indirect or non-specific. To verify the interaction directly ina cell-free system, we generated an Fc fusion of contactin 4 andassayed its binding to AP-APPs�. To measure affinity, bindingcurves were generated by varying the concentration of AP fusionprotein. To investigate specificity, we similarly tested the remainingfive contactins and another APP family protein, APLP1 (Fig. 4). Theresults revealed binding of APP to contactins 3 and 4. This fits withthe homology relationships, as contactins 3 and 4 are the mostclosely related pair within the contactin family (Ogawa et al., 1996).APLP1 bound contactins 3 and 4, and also contactin 5. No saturablebinding was detected in the other pairs tested, suggesting that if thereis an interaction, the affinity is low. The calculated dissociationconstant (KD) for APP and contactin 4 was 17 nM, whereas otherKDs ranged from 22 to 35 nM (Fig. 4B-F).

To dissect which regions of the molecules were involved, deletionconstructs were analyzed. In contactin 3 or 4, the fibronectindomains were found to be sufficient for binding. This is striking, asmost other interactions of IgCAMs are mediated by the Ig-likedomains. In APP, amino acids 18-205 were sufficient for binding(Fig. 5A,D). The localization of the binding domain to the N-terminal portion of APP, and the observation that contactins 3 and 4are GPI-anchored proteins, are consistent with these proteins beingpartly or entirely responsible for what we termed ‘binding pattern 2’in embryonic chick brain (see Fig. 2).

APP interacts with NgCAMSince our PI-PLC experiments (Fig. 2D,E) suggested the presenceof at least one non-GPI-anchored APP binding partner in tecta, wegenerated a list of further candidate APP binding partners byidentifying tectal cell-surface proteins of relevant molecularweights. Chick tecta were surface labeled with biotin, biotinylatedproteins were purified and separated by SDS-PAGE, and bandscorresponding to the molecular weights of the biotinylated bandsseen in Fig. 3A were analyzed by tandem mass spectrometry. Thesequences of the identified proteins were analyzed fortransmembrane domains or signal peptides to confirm likelyextracellular expression. The candidate proteins identified in thisway were CHL1, contactin 1, NCAM, neogenin, neurofascin,NgCAM, NrCAM, prominin-like 2 and tenascin.

To test these candidate proteins for APP association, weperformed a crosslinker-based co-IP as in Fig. 3, then usedantibodies against candidate proteins for western blot detection. Ofthe antibodies tested (see Materials and methods), only two, bothdirected against NgCAM, recognized bands in immunoprecipitatesof AP-APPs�, but not of the AP control (Fig. 5B). The primary banddetected by both NgCAM antibodies (Fig. 5B) appears tocorrespond well to the upper biotinylated band marked in Fig. 3A.Additional specific bands are also likely to be due to NgCAM;species of 80, 136, 190 and 210 kDa have been reported, with thepredominant 80 and 136 kDa forms arising by proteolysis (Burgoonet al., 1991), and these are likely to correspond to the predominant

RESEARCH ARTICLE Development 135 (6)

Fig. 2. Two different domains of APP exhibitdistinct binding properties. AP in situs on E11.5-12.5 brains. Ventral views. Arrowheads indicateolfactory bulbs. Arrows indicate optic tecta.(A) Binding of AP-APP deletion series (numberedaccording to APP695). Complete AP-APPs� is AP-APP(18-612). Binding pattern 1 refers to prominentlystriated staining specifically in the anterior tectum atE9.5. Binding pattern 2 refers to widespread stainingof the brain, including throughout the tectum, andprominent olfactory bulb staining, at E11.5-12.5.Colored bars indicate regions of APP with strongbinding in patterns 1 (green) and 2 (blue). (B,C) AP-APP(199-345) gave strong tectal staining. AP-APP(199-293) also bound to RGC axons in tecta,though less strongly. (D,E) PI-PLC or mock treatmentof brains, then AP-APPs� staining. PI-PLC treatmenteliminated most olfactory bulb staining and somebroader staining. (F,G) AP-APP(18-205) and AP-APLP1 exhibit widespread binding including in thetectum, with particular prominence in olfactory bulb.(H) Diagram of chick brain, ventral view.

DEVELO

PMENT

bands detected here (asterisks in Fig. 5B). Similar crosslinker-basedaffinity purifications using AP-APP(199-345) as a probe revealedthat NgCAM does co-precipitate with APP amino acids 199-345(Fig. 5C), the domain which produces binding pattern 1 (Fig. 2).

Contactin 4 and NgCAM modulate CTF� levelsProteolytic processing as an early step in putative APP signaltransduction has been widely proposed and studied (Chan and Jan,1998; Cao and Sudhof, 2004; Hass and Yankner, 2005). Wetherefore examined the effect of contactin 4 and NgCAM on APPprocessing. In cells transfected with APP-HA, co-expression ofcontactin 4- or NgCAM-fusion proteins resulted in increased levelsof a small HA-tagged fragment (Fig. 5E), identified as APP CTF�(C-terminal fragment �) by apparent molecular weight (Fig. 5F). Wealso saw increased levels of APP-HA, suggesting that contactin 4and NgCAM can enhance the stability or expression of full-lengthAPP. The increase in CTF� might be secondary to the increase infull-length APP, but because in some cases the increase in CTF�appeared greater than the increase in full-length APP, regulation of�-site cleavage might also be involved. Although co-transfection ofsoluble tagged forms of contactin 4 or NgCAM with APP-HAgenerally increased CTF� levels up to several fold, in a smallernumber of experiments, it resulted in an equally dramatic decreasein CTF� levels, often with an accompanying decrease in full-lengthAPP. Although we do not know the reason for these differing results,it might be related to factors such as protein concentration or cellularstate, which can affect positive versus negative responses to otherextracellular signaling molecules (Song et al., 1998; Hansen et al.,2004). In any case, these experiments demonstrate that the presenceof contactin 4 or NgCAM can modulate CTF� levels.

Expression in the visual systemFor a binding interaction to have biological significance, theinteracting proteins should have in vivo expression patterns thatallow them to interact. Since APP binds prominently to retinal axonsin the tectum (Fig. 1), we assessed expression of APP, contactins 3and 4 and NgCAM in the retinotectal system by in situ hybridizationin E11.5 chick embryos. In the retina, all four genes were expressedprominently in the RGC layer, which is the source of axons thatproject to the tectum (Fig. 6A-E,K-N). Contactin 4 was also evidentin other retinal layers. In the tectum, all four genes were expressed

in multiple layers, including superficial layers through which RGCaxons navigate (Fig. 6F-J,O-R). APP protein immunolocalizationrevealed prominent staining of the layers containing RGC axons inthe retina and tectum, as well as staining in other tectal layers (Fig.6S-Z). Along with previous reports of NgCAM expression(Lemmon and McLoon, 1986; Stoker et al., 1995; Yamagata et al.,1995; Rager et al., 1996) and our results showing that AP-APPs�binds to RGC axons in the tectum (Fig. 1), the expression patternsindicate that these four proteins are suitably placed for interactionsinvolving axon-axon or axon-target contacts in the developingretinotectal system.

Functional interactions in RGC axon outgrowthOur analyses of binding interactions and expression patternsindicated that APP, NgCAM and contactins might participate inretinal axon development, so we next tested for functional effects onRGC axons. Retinal explants cultured on NgCAM-Fc substrateshowed substantial RGC axon outgrowth, as reported previously(Doherty et al., 1995; Morales et al., 1996), whereas no outgrowthwas seen on substrate coated with AP-APPs� or contactin 4-Fc (datanot shown). Since APPs� was previously reported to promoteneurite growth in other cell types (Mattson, 1997), we tested furtherand found that outgrowth on NgCAM was enhanced by either AP-APPs� (P=0.003) or AP-APP(18-205) (P=0.024) (Fig. 7A-D).Similar results were obtained by quantitating axon number (Fig. 7D-F) or axon extension (see Fig. S1A-C in the supplementarymaterial). We next tested whether this effect of APP is specific forNgCAM, as opposed to a more generalized effect of APP,potentially unrelated to NgCAM. Our results showed that APPs�specifically promoted NgCAM-supported outgrowth, but notoutgrowth on other substrates such as laminin (Fig. 7E),demonstrating a specific functional interaction between APP andNgCAM in regulating axon growth.

The observation that the N-terminal domain of APP, which bindscontactins 3 and 4, was sufficient to potentiate NgCAM-dependentoutgrowth, suggested a model in which axonal contactin 3 or 4 actsas a receptor for APP. This model predicts that soluble contactin 4could act as a dominant-negative and block signaling throughaxonally expressed contactin 3 or 4. We therefore tested contactin 4-Fc, and found that it did indeed inhibit outgrowth, reducing levels ofoutgrowth on AP-APPs� plus NgCAM (P=0.011) to levels seen on

1193RESEARCH ARTICLEAPP interaction with contactins and NgCAM

Fig. 3. Crosslinker-basedaffinity purification of APP-interacting proteins from tecta.AP probes were crosslinked tosurface-biotinylated tecta, thenimmunoprecipitated from lysatesto identify associated proteins.Crosslinker was cleaved before gelanalysis. (A) Western blot usingNeutrAvidin-HRP to detectsurface-biotinylated co-precipitated proteins. Asterisksmark the two predominant bands.(B-G) 2D gels for large-scale co-precipitation. Western blots forbiotinylated proteins (B,E) werecompared with silver-stained gels(C,D,F,G). Arrows indicate thepredominant biotinylated proteins in B and corresponding locations in the silver-stained gels. This region was excised from gels in C and D fortandem mass spectrometry. Arrowhead indicates an additional spot specific for the AP-APPs� co-IP: mass spectrometry identified severalproteins, but these lacked obvious signal peptide or transmembrane domains and were not characterized further.

DEVELO

PMENT

1194

NgCAM alone (Fig. 7E). Contactin 4-Fc also reduced growth onNgCAM alone, but, importantly, showed no effect on laminin-dependent growth of RGC axons (Fig. 7E,F), thereby demonstratingspecificity. We further tested the model by examining axonoutgrowth from retinas infected with virus expressing shRNA thattargets contactin 4. Explants expressing shRNA against contactin 4exhibited less axon growth than control explants, when grown onAP-APPs� plus NgCAM (P=0.021), whereas outgrowth on lamininwas not significantly affected (Fig. 7G-K). Thus, data from twoindependent experimental approaches support a role for contactin 4in the response by RGCs axons to NgCAM plus AP-APPs�.

DISCUSSIONAPP has been studied extensively for its role in Alzheimer’s disease.Genetic studies show that APP functions in neural development, butspecific mechanisms of APP action in vertebrate development haveremained unclear. To find developmentally relevant binding partnersfor APP, we initially undertook a series of biochemical studies indeveloping chick brain, which revealed interactions of the APPextracellular domain with contactin 3, contactin 4 and NgCAM.

Evidence for a ligand-receptor relationship can be provided byshowing a binding interaction, expression in suitable places tointeract and a functional interaction. In keeping with this, in additionto the binding interaction demonstrated by our molecular studies, we

find that all these molecules are expressed in the developingretinotectal system, and that APPs� and contactin 4 can specificallymodulate NgCAM-dependent axon outgrowth, demonstratingfunctional interactions among these proteins that can induce changesin cellular behavior.

Binding of APP family members to proteins of thecontactin and L1CAM familiesOur studies show that APP binds both contactin 3 and 4. APLP1 wasfound to bind contactins 3 and 4, and also contactin 5. These resultsthus demonstrate a set of promiscuous binding interactions betweenthe APP and contactin families, although binding was not seen in allpairwise combinations, thereby indicating a degree of specificity.Although our functional studies have so far focused on APP andcontactin 4, it is likely that interaction among other APP andcontactin family members will also have functional consequences.

Binding of APP to contactins 3 and 4 is a direct interaction thatcan be observed in a cell-free system. On contactin 4, the APP-binding site localizes to the FN-like domains, which could leave theIg domains of contactin free to interact with other binding partners.On APP, the contactin-binding site is at the N-terminus, involvingamino acids 18-205. This corresponds essentially to the E1 domainof APPs� (Daigle and Li, 1993), which has been implicated in manybiological processes, including neural stem cell proliferation

RESEARCH ARTICLE Development 135 (6)

Fig. 4. APP and APLP1 bind to specificcontactin family members. (A) APfusion proteins were tested for binding tocontactin-Fc fusion proteins immobilizedon Protein A beads. AP activity retainedafter washing beads is shown. APP boundprominently to contactins 3 and 4. APLP1bound to contactins 3, 4 and 5. Somenon-saturable binding indicating weak ornon-specific binding was also seen withcontactin 1-Fc (data not shown).(B-F) Saturation curves (insets) weregenerated by varying the concentration ofAP fusion protein. Lines in correspondingScatchard plots were generated by leastsquares fitting. KDs: contactin 4-APP, 17nM; contactin 4-APLP1, 28 nM; contactin3-APP, 22 nM; contactin 3-APLP1, 35 nM;and contactin 5-APLP1, 32 nM.

DEVELO

PMENT

(Ohsawa et al., 1999), synaptogenesis (Morimoto et al., 1998) andneurite growth (Small et al., 1994; Ohsawa et al., 1997), consistentwith the possibility that interactions with contactins could beinvolved in these biological processes.

APP also associated in our co-precipitation experiments withNgCAM. We have so far not been able to show direct binding incell-free assays, which could have various interpretations: theassociation could be indirect, or it might require additionalcomponents, or direct binding can occur but was not evident owingto technical aspects of the assay. Interestingly, the interaction withNgCAM does not seem to be mediated by contactins in any simpleway, as NgCAM can be crosslinked to AP-APP(199-345), aconstruct without the binding domain for contactins. The presenceof distinct domains in APP that interact with NgCAM versuscontactins suggests a model in which the three proteins functiontogether in a three-way complex. Previous studies have shown thatL1CAM family members interact with contactin Ig domains(Brummendorf and Rathjen, 1996), so our finding that APP interactswith contactin FN domains could be consistent with simultaneousor cooperative binding in a multimeric complex.

APP processingAPP cleavage can occur along two pathways. In the amyloidogenicpathway, cleavage by �- and �-secretases generates A�, implicatedin Alzheimer’s disease. In the non-amyloidogenic pathway, cleavage

by �-secretase precludes the formation of A�, suggesting thepotential to protect against Alzheimer’s disease. The non-amyloidogenic pathway has also been implicated in biologicalsignaling because it generates the extracellular APPs� fragment thatcan regulate cell behavior when added to cell cultures, and because�-cleavage has been proposed to generate a signal within the APP-bearing cell. We were therefore interested to find that contactin 4 andNgCAM can alter the level of CTF�, the C-terminal �-cleavageproduct, by several fold. Although we have not characterized thespecific mechanism (regulation of APP cleavage, stability,subcellular localization, etc.), our experiments show effects on thelevel of CTF�, and any net change in the level of CTF� is abiochemical signaling event that may influence downstreampathways. Further studies would be required to determine thebiologically relevant signaling mechanisms, and whether theinteractions identified here could modulate A� production withtherapeutic relevance.

Functional interactions in regulating RGC axongrowthFurther evidence for interactions among these proteins comesfrom our functional studies of RGC axon outgrowth. APPs�specifically potentiated NgCAM-dependent, but not laminin-dependent, axon outgrowth, demonstrating a specific functionalinteraction between APPs� and NgCAM. Interfering with

1195RESEARCH ARTICLEAPP interaction with contactins and NgCAM

Fig. 5. Characterization ofinteractions of APP with contactinsand NgCAM. (A) AP-fused APP domainswere tested for binding to Fc-fusedcontactin 3 or 4 domains [forconcentrations, see Materials andmethods; for domain selection see Raderet al. (Rader et al., 1996)]. Bindingactivity localizes to the fibronectin (FN)domains of contactins, and to the N-terminal domain of APP. (B) APPassociation with NgCAM. Western blotswith antibodies against NgCAM afterimmunoprecipitating for AP tag.Asterisks mark major bands specificallyco-immunoprecipitated with AP-APPs�,consistent with published molecularweights of NgCAM. (C) Amino acids199-345 of APP are sufficient forassociation with NgCAM. Non-cleavablecrosslinker BS3 used here to examine netmolecular weight of immunoprecipitatedcomplexes. Resulting spread of signalmight indicate the presence ofadditional molecules in the crosslinkedNgCAM-APP complexes. (D) Model forinteractions among proteins. N-terminaldomain of APP (amino acids 18-205,‘E1’) binds directly to fibronectindomains of contactin 4, whereas aminoacids 199-345 of APP interact, directlyor indirectly, with NgCAM. Amino acids199-345 of APP encompass the acidicdomain (oval, ‘A’) and the N-terminalportion of the central APP domain,termed E2, which includes the RERMSpeptide previously implicated in APPs� function (Reinhard et al., 2005). (E) Co-transfection of APP-HA and indicated constructs, followed byanti-HA western blot. Contactin 4-Fc or NgCAM-Fc constructs increased the CTF� level compared with Fc control. (F) Co-migration with anartificial CTF� polypeptide confirms the identity of the APP cleavage fragment observed after co-expression with contactin 4-AP.

DEVELO

PMENT

1196

contactin 4 by two independent methods, using a putativedominant negative, or using shRNA against contactin 4, inhibitedoutgrowth on NgCAM and APPs�, but not on laminin, againdemonstrating specific functional interactions.

If interactions among APP, NgCAM and contactin 4 are toaffect RGC axon behavior in vivo, these genes must be expressedat a relevant time and location. RNA in situ hybridization showedthat all three genes are expressed in both the RGC layer of theretina and in the optic tectum at the time of retinotectaldevelopment. To investigate functional effects on RGC axons, weused an assay of axon outgrowth, although it is worth noting thatmolecules with activity in axon outgrowth assays can havebiological roles in axon growth, guidance or synaptogenesis.Since none of these molecules was seen in an obvious gradient,they are presumably not acting as positional mapping labels, butthey might function in other axon-axon or axon-target interactionsthat are involved in retinotectal development (McLaughlin andO’Leary, 2005; Flanagan, 2006). Indeed, genetic studies havealready shown that the mammalian NgCAM homolog, L1CAM,is required for normal development of the retinotectal map

(Demyanenko and Maness, 2003). In view of the interactions seenhere, it will ultimately be interesting to further test the roles of theL1CAM, APP and contactin families in vivo.

Since APPs� and NgCAM stimulated RGC axon growth whenpresented together in trans, and as they can interact physically, alikely model is that they can function as a co-ligand complex, jointlyinteracting with and activating a receptor. Since the domain of APPthat interacts with contactin 4 is sufficient to promote axon growth,because both contactin 4-Fc and RNAi against contactin 4 inhibitedgrowth on NgCAM and APPs�, and because published studies haveimplicated contactin 2 as a co-receptor in NgCAM-promoted neuriteoutgrowth (Buchstaller et al., 1996), our data support the model thataxonal contactin 4 acts here by mediating a response to moleculeson the substratum. Taken together, our functional studies of axongrowth in vitro lead to a working model (Fig. 7L) in which contactin4 would act as an axonal receptor or co-receptor for an APPs�-NgCAM co-ligand complex. However, although our experimentsclearly support functional interactions among these proteins, furtherstudies would be required to investigate exactly which cis and transinteractions may occur in a biological context.

RESEARCH ARTICLE Development 135 (6)

Fig. 6. Expression of APP, NgCAM, contactin 4 andcontactin 3 in the developing visual system. RNA in situhybridization in sagittal sections of E11.5 chick heads.(A-E) Retina. Each antisense probe detected RNA expressionin the RGC layer (arrow). Pigmented epithelium (dark brownline) bounds the retina opposite the RGC layer. (F-J) Eachantisense probe detected RNA expression in multiple layersof the tectum. Top, anterior extreme; bottom, posteriorextreme; open arrowheads, stratum opticum as identified byGAP43 immunolabeling. (K-Z) Co-staining using RNA in situhybridization (K-R) or immunohistochemistry (S-Z) for APP(green), DAPI nuclear stain (blue) and immunohistochemistryfor GAP43 (red). Solid arrow, RGC layer; open arrow, retinaloptic fiber layer; solid arrowhead, pigmented epithelium;open arrowheads, tectal stratum opticum as identified byGAP43 immunolabeling. Scale bars: 500 �m in A-J; 100 �min K-V; 200 �m in W-Z.

DEVELO

PMENT

APP has been widely proposed to function as both a receptor anda ligand. In principle, these two functions could involve either thesame or different molecular interactions. Our results showing effectsof contactin 4 and NgCAM on APP processing could be consistentwith a receptor function for APP. Meanwhile, our studies of RGCaxon outgrowth support a model in which APPs� and NgCAM canact together as ligands. These models are entirely consistent,assuming formation of a complex that can have multiple signalingoutputs. Precedent for such a model comes from signalingcomplexes, such as ephrins and Eph receptors, which can interact incis and trans, and signal bidirectionally (Kullander and Klein, 2002;Flanagan, 2006).

APP, contactins and L1CAMs in development anddiseaseInterestingly, there are a number of biological and pathologicalprocesses in which both APP and IgCAMs related to NgCAM orcontactins have been implicated. For example, mice with a disruptedAPP gene exhibit defects in corpus callosum formation (Magara etal., 1999) reminiscent of the defects seen in patients with CRASHsyndrome, which is caused by mutations in L1CAM, the humanhomolog of NgCAM (Fransen et al., 1995). Overexpression ofFASII in Drosophila results in an increased number ofneuromuscular synaptic boutons, which is dependent ondownstream function of the APP homolog APPL (Ashley et al.,2005). FASII is the Drosophila homolog of NCAM, which is distinctfrom, but closely related to, the Drosophila homologs of the

contactins (CONT) and the L1CAMs (Neuroglian) (Vaughn andBjorkman, 1996; Hortsch, 2000; Faivre-Sarrailh et al., 2004), sothese results might reflect an underlying evolutionary conservationof interactions.

More generally, multiple studies implicate members of theAPP, contactin and L1CAM families in the development ofneural connectivity in vertebrates. APPs� can affect neuriteoutgrowth, synaptogenesis, and synaptic plasticity (Mattson,1997; Turner et al., 2003). Contactins and L1CAMs have beenextensively studied for their roles in neurite outgrowth(Sonderegger, 1997; Kamiguchi et al., 1998; Falk et al., 2002),and contactin 1 and L1CAM have been implicated in synapseformation or plasticity (Hoffman, 1998; Murai et al., 2002;Saghatelyan et al., 2004).

A ligand for APP might also be expected to affect progressionof Alzheimer’s disease, either by regulating functions of APP thatcontrol cell behavior, or by modulating the processing of APP toA�. Consistent with this, CNTN4 maps to chromosome 3p26,only 1.1 Mb from the D3S2387 marker, which was reported tohave suggestive genetic linkage to Alzheimer’s disease (Blackeret al., 2003); intriguingly, the only other identified genes asclosely located to this marker are CNTN6 (encoding contactin 6)and CHL1 (encoding the L1CAM family protein, close homologof L1). The work described here identifies molecular andfunctional interactions of APP with contactin and L1CAM familyproteins, which might have general roles in neural developmentand disease.

1197RESEARCH ARTICLEAPP interaction with contactins and NgCAM

Fig. 7. Interactions among NgCAM,contactin 4 and APPs� in RGC axonoutgrowth. Retinal strips cultured on differentsubstrates before axon imaging. (A-D) APPs�and APP(18-205) both enhanced outgrowth onNgCAM. AP, n=12; other conditions, n=6.(E) APPs� enhanced NgCAM-dependent, butnot laminin-dependent, axon outgrowth. n=6.(F) Contactin 4-Fc inhibitied NgCAM-dependent, but not laminin-dependent, axonoutgrowth. n=10. Protein coating was varied indifferent experiments; the high basal outgrowthhere allowed for robust quantitation of theinhibitory effect of contactin 4-Fc.(G-K) Contactin 4 shRNA in replication-competent retrovirus inhibited axon outgrowthon NgCAM+AP-APPs�, but not on laminin.Laminin, n=11 (shRNA), n=11 (control virus);NgCAM+APPs�, n=21 (shRNA), n=19 (controlvirus). (L) Model of APPs�, NgCAM andcontactin 4 function based on the assays ofRGC axon outgrowth shown here. Other cis andtrans interactions might also occur. See text fordetails. *, P<0.025 by Student’s t-test. Errorbars, s.e.m. Scale bars: 300 �m.

DEVELO

PMENT

1198

We thank Edna Sun, Masaru Nakamoto, Jonaki Sen, Mark Emerson, SanjivHarpavat, and Samira Pontes for advice and help with experiments; RossTomaino and Steve Gygi for performing and interpreting the massspectrometry analysis; Peter Sonderegger, Josh Sanes and Fritz Rathjen forantibodies and plasmids; David Van Vactor, Connie Cepko, Alan Tenney,Xinmin Li, David Feldheim, Qiang Lu, Nicolas Preitner, Dan Nowakowski andother members of the Flanagan and Van Vactor laboratories for discussions;and Dennis Selkoe and Rudolph Tanzi for comments on the manuscript. Thiswork was supported by grants EY11559 and HD29417 from the NationalInstitutes of Health (to J.G.F.) and by a postdoctoral fellowship from the AlfredBenzon Foundation, Denmark (to R.E.).

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/135/6/1189/DC1

ReferencesAshley, J., Packard, M., Ataman, B. and Budnik, V. (2005). Fasciclin II signals

new synapse formation through amyloid precursor protein and the scaffoldingprotein dX11/Mint. J. Neurosci. 25, 5943-5955.

Beher, D., Hesse, L., Masters, C. L. and Multhaup, G. (1996). Regulation ofamyloid protein precursor (APP) binding to collagen and mapping of the bindingsites on APP and collagen type I. J. Biol. Chem. 271, 1613-1620.

Blacker, D., Bertram, L., Saunders, A. J., Moscarillo, T. J., Albert, M. S.,Wiener, H., Perry, R. T., Collins, J. S., Harrell, L. E., Go, R. C. et al. (2003).Results of a high-resolution genome screen of 437 Alzheimer’s disease families.Hum. Mol. Genet. 12, 23-32.

Brummendorf, T. and Rathjen, F. G. (1996). Structure/function relationships ofaxon-associated adhesion receptors of the immunoglobulin superfamily. Curr.Opin. Neurobiol. 6, 584-593.

Buchstaller, A., Kunz, S., Berger, P., Kunz, B., Ziegler, U., Rader, C. andSonderegger, P. (1996). Cell adhesion molecules NgCAM and axonin-1 formheterodimers in the neuronal membrane and cooperate in neurite outgrowthpromotion. J. Cell Biol. 135, 1593-1607.

Burgoon, M. P., Grumet, M., Mauro, V., Edelman, G. M. and Cunningham, B.A. (1991). Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily. J. Cell Biol.112, 1017-1029.

Caille, I., Allinquant, B., Dupont, E., Bouillot, C., Langer, A., Muller, U. andProchiantz, A. (2004). Soluble form of amyloid precursor protein regulatesproliferation of progenitors in the adult subventricular zone. Development 131,2173-2181.

Cao, X. and Sudhof, T. C. (2001). A transcriptionally [correction of transcriptively]active complex of APP with Fe65 and histone acetyltransferase Tip60. Science293, 115-120.

Cao, X. and Sudhof, T. C. (2004). Dissection of amyloid-beta precursor protein-dependent transcriptional transactivation. J. Biol. Chem. 279, 24601-24611.

Catalano, S. M., Dodson, E. C., Henze, D. A., Joyce, J. G., Krafft, G. A. andKinney, G. G. (2006). The role of amyloid-beta derived diffusible ligands(ADDLs) in Alzheimer’s disease. Curr. Top. Med. Chem. 6, 597-608.

Chan, Y. M. and Jan, Y. N. (1998). Roles for proteolysis and trafficking in notchmaturation and signal transduction. Cell 94, 423-426.

Chang, S., Rathjen, F. G. and Raper, J. A. (1990). Neurite outgrowth promotingactivity of G4 and its inhibition by monoclonal antibodies. J. Neurosci. Res. 25,180-186.

Chen, C. D., Oh, S. Y., Hinman, J. D. and Abraham, C. R. (2006). Visualizationof APP dimerization and APP-Notch2 heterodimerization in living cells usingbimolecular fluorescence complementation. J. Neurochem. 97, 30-43.

Connelly, M. A., Grady, R. C., Mushinski, J. F. and Marcu, K. B. (1994). PANG,a gene encoding a neuronal glycoprotein, is ectopically activated byintracisternal A-type particle long terminal repeats in murine plasmacytomas.Proc. Natl. Acad. Sci. USA 91, 1337-1341.

Daigle, I. and Li, C. (1993). apl-1, a Caenorhabditis elegans gene encoding aprotein related to the human beta-amyloid protein precursor. Proc. Natl. Acad.Sci. USA 90, 12045-12049.

Demyanenko, G. P. and Maness, P. F. (2003). The L1 cell adhesion molecule isessential for topographic mapping of retinal axons. J. Neurosci. 23, 530-538.

Dijkhuizen, T., van Essen, T., van der Vlies, P., Verheij, J. B., Sikkema-Raddatz, B., van der Veen, A. Y., Gerssen-Schoorl, K. B., Buys, C. H. andKok, K. (2006). FISH and array-CGH analysis of a complex chromosome 3aberration suggests that loss of CNTN4 and CRBN contributes to mentalretardation in 3pter deletions. Am. J. Med. Genet. A 140, 2482-2487.

Doherty, P., Williams, E. and Walsh, F. S. (1995). A soluble chimeric form of theL1 glycoprotein stimulates neurite outgrowth. Neuron 14, 57-66.

Faivre-Sarrailh, C., Banerjee, S., Li, J., Hortsch, M., Laval, M. and Bhat, M. A.(2004). Drosophila contactin, a homolog of vertebrate contactin, is required forseptate junction organization and paracellular barrier function. Development131, 4931-4942.

Falk, J., Bonnon, C., Girault, J. A. and Faivre-Sarrailh, C. (2002). F3/contactin,a neuronal cell adhesion molecule implicated in axogenesis and myelination.Biol. Cell 94, 327-334.

Fassa, A., Mehta, P. and Efthimiopoulos, S. (2005). Notch 1 interacts with theamyloid precursor protein in a Numb-independent manner. J. Neurosci. Res. 82,214-224.

Fernandez, T., Morgan, T., Davis, N., Klin, A., Morris, A., Farhi, A., Lifton, R.P. and State, M. W. (2004). Disruption of contactin 4 (CNTN4) results indevelopmental delay and other features of 3p deletion syndrome. Am. J. Hum.Genet. 74, 1286-1293.

Fischer, D. F., van Dijk, R., Sluijs, J. A., Nair, S. M., Racchi, M., Levelt, C. N.,van Leeuwen, F. W. and Hol, E. M. (2005). Activation of the Notch pathway inDown syndrome: cross-talk of Notch and APP. FASEB J. 19, 1451-1458.

Flanagan, J. G. (2006). Neural map specification by gradients. Curr. Opin.Neurobiol. 16, 59-66.

Flanagan, J. G. and Cheng, H. J. (2000). Alkaline phosphatase fusion proteins formolecular characterization and cloning of receptors and their ligands. Meth.Enzymol. 327, 198-210.

Flanagan, J. G., Cheng, H. J., Feldheim, D. A., Hattori, M., Lu, Q. andVanderhaeghen, P. (2000). Alkaline phosphatase fusions of ligands orreceptors as in situ probes for staining of cells, tissues, and embryos. Meth.Enzymol. 327, 19-35.

Fotinopoulou, A., Tsachaki, M., Vlavaki, M., Poulopoulos, A., Rostagno, A.,Frangione, B., Ghiso, J. and Efthimiopoulos, S. (2005). BRI2 interacts withamyloid precursor protein (APP) and regulates amyloid beta (Abeta) production.J. Biol. Chem. 280, 30768-30772.

Fransen, E., Lemmon, V., Van Camp, G., Vits, L., Coucke, P. and Willems, P. J.(1995). CRASH syndrome: clinical spectrum of corpus callosum hypoplasia,retardation, adducted thumbs, spastic paraparesis and hydrocephalus due tomutations in one single gene, L1. Eur. J. Hum. Genet. 3, 273-284.

Goldberg, S. (1974). Studies on the mechanics of development of the visualpathways in the chick embryo. Dev. Biol. 36, 24-43.

Hansen, M. J., Dallal, G. E. and Flanagan, J. G. (2004). Retinal axon response toephrin-as shows a graded, concentration-dependent transition from growthpromotion to inhibition. Neuron 42, 717-730.

Hardy, J. and Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer’s disease:progress and problems on the road to therapeutics. Science 297, 353-356.

Hass, M. R. and Yankner, B. A. (2005). A gamma-secretase-independentmechanism of signal transduction by the amyloid precursor protein. J. Biol.Chem. 280, 36895-36904.

Hebert, S. S., Serneels, L., Tolia, A., Craessaerts, K., Derks, C., Filippov, M. A.,Muller, U. and De Strooper, B. (2006). Regulated intramembrane proteolysisof amyloid precursor protein and regulation of expression of putative targetgenes. EMBO Rep. 7, 739-745.

Herms, J., Anliker, B., Heber, S., Ring, S., Fuhrmann, M., Kretzschmar, H.,Sisodia, S. and Muller, U. (2004). Cortical dysplasia resembling human type 2lissencephaly in mice lacking all three APP family members. EMBO J. 23, 4106-4115.

Ho, A. and Sudhof, T. C. (2004). Binding of F-spondin to amyloid-beta precursorprotein: a candidate amyloid-beta precursor protein ligand that modulatesamyloid-beta precursor protein cleavage. Proc. Natl. Acad. Sci. USA 101, 2548-2553.

Hoe, H. S., Wessner, D., Beffert, U., Becker, A. G., Matsuoka, Y. and Rebeck,G. W. (2005). F-spondin interaction with the apolipoprotein E receptor ApoEr2affects processing of amyloid precursor protein. Mol. Cell. Biol. 25, 9259-9268.

Hoffman, K. B. (1998). The relationship between adhesion molecules andneuronal plasticity. Cell. Mol. Neurobiol. 18, 461-475.

Hortsch, M. (1996). The L1 family of neural cell adhesion molecules: old proteinsperforming new tricks. Neuron 17, 587-593.

Hortsch, M. (2000). Structural and functional evolution of the L1 family: are fouradhesion molecules better than one? Mol. Cell. Neurosci. 15, 1-10.

Kamiguchi, H., Hlavin, M. L. and Lemmon, V. (1998). Role of L1 in neuraldevelopment: what the knockouts tell us. Mol. Cell. Neurosci. 12, 48-55.

Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L.,Grzeschik, K. H., Multhaup, G., Beyreuther, K. and Muller-Hill, B. (1987).The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surfacereceptor. Nature 325, 733-736.

Kerr, M. L. and Small, D. H. (2005). Cytoplasmic domain of the beta-amyloidprotein precursor of Alzheimer’s disease: function, regulation of proteolysis, andimplications for drug development. J. Neurosci. Res. 80, 151-159.

Kimberly, W. T., Zheng, J. B., Guenette, S. Y. and Selkoe, D. J. (2001). Theintracellular domain of the beta-amyloid precursor protein is stabilized by Fe65and translocates to the nucleus in a notch-like manner. J. Biol. Chem. 276,40288-40292.

Kinoshita, A., Whelan, C. M., Smith, C. J., Berezovska, O. and Hyman, B. T.(2002). Direct visualization of the gamma secretase-generated carboxyl-terminaldomain of the amyloid precursor protein: association with Fe65 andtranslocation to the nucleus. J. Neurochem. 82, 839-847.

Kounnas, M. Z., Moir, R. D., Rebeck, G. W., Bush, A. I., Argraves, W. S., Tanzi,R. E., Hyman, B. T. and Strickland, D. K. (1995). LDL receptor-related protein,

RESEARCH ARTICLE Development 135 (6)

DEVELO

PMENT

a multifunctional ApoE receptor, binds secreted beta-amyloid precursor proteinand mediates its degradation. Cell 82, 331-340.

Kullander, K. and Klein, R. (2002). Mechanisms and functions of Eph and ephrinsignalling. Nat. Rev. Mol. Cell Biol. 3, 475-486.

Lemmon, V. and McLoon, S. C. (1986). The appearance of an L1-like molecule inthe chick primary visual pathway. J. Neurosci. 6, 2987-2994.

Magara, F., Muller, U., Li, Z. W., Lipp, H. P., Weissmann, C., Stagljar, M. andWolfer, D. P. (1999). Genetic background changes the pattern of forebraincommissure defects in transgenic mice underexpressing the beta-amyloid-precursor protein. Proc. Natl. Acad. Sci. USA 96, 4656-4661.

Majercak, J., Ray, W. J., Espeseth, A., Simon, A., Shi, X. P., Wolffe, C., Getty,K., Marine, S., Stec, E., Ferrer, M. et al. (2006). LRRTM3 promotes processingof amyloid-precursor protein by BACE1 and is a positional candidate gene forlate-onset Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 103, 17967-17972.

Maness, P. F. and Schachner, M. (2007). Neural recognition molecules of theimmunoglobulin superfamily: signaling transducers of axon guidance andneuronal migration. Nat. Neurosci. 10, 19-26.

Matsuda, S., Giliberto, L., Matsuda, Y., Davies, P., McGowan, E., Pickford, F.,Ghiso, J., Frangione, B. and D’Adamio, L. (2005). The familial dementia BRI2gene binds the Alzheimer gene amyloid-beta precursor protein and inhibitsamyloid-beta production. J. Biol. Chem. 280, 28912-28916.

Mattson, M. P. (1997). Cellular actions of beta-amyloid precursor protein and itssoluble and fibrillogenic derivatives. Physiol. Rev. 77, 1081-1132.

McLaughlin, T. and O’Leary, D. D. (2005). Molecular gradients and developmentof retinotopic maps. Annu. Rev. Neurosci. 28, 327-355.

Morales, G., Sanchez-Puelles, J. M., Schwarz, U. and de la Rosa, E. J. (1996).Synergistic neurite-outgrowth promoting activity of two related axonal proteins,Bravo/Nr-CAM and G4/Ng-CAM in chicken retinal explants. Eur. J. Neurosci. 8,1098-1105.

Morimoto, T., Ohsawa, I., Takamura, C., Ishiguro, M. and Kohsaka, S. (1998).Involvement of amyloid precursor protein in functional synapse formation incultured hippocampal neurons. J. Neurosci. Res. 51, 185-195.

Multhaup, G. (1994). Identification and regulation of the high affinity binding siteof the Alzheimer’s disease amyloid protein precursor (APP) toglycosaminoglycans. Biochimie 76, 304-311.

Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev.Biol. 228, 151-165.

Murai, K. K., Misner, D. and Ranscht, B. (2002). Contactin supports synapticplasticity associated with hippocampal long-term depression but notpotentiation. Curr. Biol. 12, 181-190.

Ogawa, J., Kaneko, H., Masuda, T., Nagata, S., Hosoya, H. and Watanabe, K.(1996). Novel neural adhesion molecules in the Contactin/F3 subgroup of theimmunoglobulin superfamily: isolation and characterization of cDNAs from ratbrain. Neurosci. Lett. 218, 173-176.

Oh, S. Y., Ellenstein, A., Chen, C. D., Hinman, J. D., Berg, E. A., Costello, C.E., Yamin, R., Neve, R. L. and Abraham, C. R. (2005). Amyloid precursorprotein interacts with notch receptors. J. Neurosci. Res. 82, 32-42.

Ohsawa, I., Takamura, C. and Kohsaka, S. (1997). The amino-terminal region ofamyloid precursor protein is responsible for neurite outgrowth in rat neocorticalexplant culture. Biochem. Biophys. Res. Commun. 236, 59-65.

Ohsawa, I., Takamura, C., Morimoto, T., Ishiguro, M. and Kohsaka, S. (1999).Amino-terminal region of secreted form of amyloid precursor protein stimulatesproliferation of neural stem cells. Eur. J. Neurosci. 11, 1907-1913.

Ohsawa, I., Takamura, C. and Kohsaka, S. (2001). Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiologicalfunction. J. Neurochem. 76, 1411-1420.

Park, J. H., Gimbel, D. A., GrandPre, T., Lee, J. K., Kim, J. E., Li, W., Lee, D. H.and Strittmatter, S. M. (2006). Alzheimer precursor protein interaction withthe Nogo-66 receptor reduces amyloid-beta plaque deposition. J. Neurosci. 26,1386-1395.

Rader, C., Kunz, B., Lierheimer, R., Giger, R. J., Berger, P., Tittmann, P., Gross,H. and Sonderegger, P. (1996). Implications for the domain arrangement ofaxonin-1 derived from the mapping of its NgCAM binding site. EMBO J. 15,2056-2068.

Rager, G., Morino, P., Schnitzer, J. and Sonderegger, P. (1996). Expression ofthe axonal cell adhesion molecules axonin-1 and Ng-CAM during thedevelopment of the chick retinotectal system. J. Comp. Neurol. 365, 594-609.

Reinhard, C., Hebert, S. S. and De Strooper, B. (2005). The amyloid-betaprecursor protein: integrating structure with biological function. EMBO J. 24,3996-4006.

Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S. andKhvorova, A. (2004). Rational siRNA design for RNA interference. Nat.Biotechnol. 22, 326-330.

Saghatelyan, A. K., Nikonenko, A. G., Sun, M., Rolf, B., Putthoff, P., Kutsche,M., Bartsch, U., Dityatev, A. and Schachner, M. (2004). Reduced GABAergictransmission and number of hippocampal perisomatic inhibitory synapses injuvenile mice deficient in the neural cell adhesion molecule L1. Mol. Cell.Neurosci. 26, 191-203.

Santiago-Garcia, J., Mas-Oliva, J., Innerarity, T. L. and Pitas, R. E. (2001).Secreted forms of the amyloid-beta precursor protein are ligands for the class Ascavenger receptor. J. Biol. Chem. 276, 30655-30661.

Schulte, D., Furukawa, T., Peters, M. A., Kozak, C. A. and Cepko, C. L. (1999).Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizesthe retina and perturbs the retinotectal map. Neuron 24, 541-553.

Selkoe, D. J. (1999). Translating cell biology into therapeutic advances inAlzheimer’s disease. Nature 399, A23-A31.

Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996). Mass spectrometricsequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858.

Small, D. H., Nurcombe, V., Reed, G., Clarris, H., Moir, R., Beyreuther, K. andMasters, C. L. (1994). A heparin-binding domain in the amyloid proteinprecursor of Alzheimer’s disease is involved in the regulation of neuriteoutgrowth. J. Neurosci. 14, 2117-2127.

Soba, P., Eggert, S., Wagner, K., Zentgraf, H., Siehl, K., Kreger, S., Lower, A.,Langer, A., Merdes, G., Paro, R. et al. (2005). Homo- and heterodimerizationof APP family members promotes intercellular adhesion. EMBO J. 24, 3624-3634.

Sonderegger, P. (1997). Axonin-1 and NgCAM as “recognition” components ofthe pathway sensor apparatus of growth cones: a synopsis. Cell Tissue Res. 290,429-439.

Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne,M. and Poo, M. (1998). Conversion of neuronal growth cone responses fromrepulsion to attraction by cyclic nucleotides. Science 281, 1515-1518.

Steinbach, J. P., Muller, U., Leist, M., Li, Z. W., Nicotera, P. and Aguzzi, A.(1998). Hypersensitivity to seizures in beta-amyloid precursor protein deficientmice. Cell Death Differ. 5, 858-866.

Stoker, A. W., Gehrig, B., Newton, M. R. and Bay, B. H. (1995). Comparativelocalisation of CRYP alpha, a CAM-like tyrosine phosphatase, and NgCAM in thedeveloping chick visual system. Brain Res. Dev. Brain Res. 90, 129-140.

Tanzi, R. E. and Bertram, L. (2005). Twenty years of the Alzheimer’s diseaseamyloid hypothesis: a genetic perspective. Cell 120, 545-555.

Turner, P. R., O’Connor, K., Tate, W. P. and Abraham, W. C. (2003). Roles ofamyloid precursor protein and its fragments in regulating neural activity,plasticity and memory. Prog. Neurobiol. 70, 1-32.

Vaughn, D. E. and Bjorkman, P. J. (1996). The (Greek) key to structures of neuraladhesion molecules. Neuron 16, 261-273.

Yamagata, M., Herman, J. P. and Sanes, J. R. (1995). Lamina-specific expressionof adhesion molecules in developing chick optic tectum. J. Neurosci. 15, 4556-4571.

Yoshihara, Y., Kawasaki, M., Tani, A., Tamada, A., Nagata, S., Kagamiyama,H. and Mori, K. (1994). BIG-1: a new TAG-1/F3-related member of theimmunoglobulin superfamily with neurite outgrowth-promoting activity. Neuron13, 415-426.

Yoshihara, Y., Kawasaki, M., Tamada, A., Nagata, S., Kagamiyama, H. andMori, K. (1995). Overlapping and differential expression of BIG-2, BIG-1, TAG-1,and F3: four members of an axon-associated cell adhesion molecule subgroup ofthe immunoglobulin superfamily. J. Neurobiol. 28, 51-69.

1199RESEARCH ARTICLEAPP interaction with contactins and NgCAM

DEVELO

PMENT